Many viruses activate the cellular NF-kappa B transcription factor as a method to express their own viral genes. However, NF-kappa B is dangerous as it induces expression of many anti-viral immune and inflammatory molecules. Thus, many viruses also produce proteins to inhibit NF-kappa B activation. Obviously, the timing, degree and pathways for NF-kappa B activation must be precisely regulated by viruses to ensure virus gene expression and immune evasion. Poxvirus infections both induce and inhibit NF-kappa B activation. However, very few individual genes have been identified as being responsible for these global effects. Even less clear is how poxviruses regulate NF-kappa B during infection to balance its beneficial and detrimental effects. Our working hypothesis is that vaccinia contains as yet undiscovered NF-kappa B inhibitors and activators, with inhibitory proteins responsible for dampening immune responses as a mechanism for virulence and activating proteins important for virus replication. Using a genetic approach, we have determined a 5.2 kilobase region of VV DNA that inhibits MVA-induced NF-kappa B activation. Known poxviral NF-kappa B inhibitors are not present in this 5.2 kb region, suggesting that VV genes are bona fide NF-kappa B inhibitors. To determine the novel NF-kappa B inhibitor(s), individual genes from this 5.2 kb region (namely the M1L, M2L and K1L genes) will be cloned and expressed independently in recombinant MVA viruses to identify NF-kappa B inhibitory activity. Using this approach, we have already identified the K1L and M2L products as NF-kappa B inhibitors (Specific Aim1). Defining the molecular mechanism used by K1L or M2L will give insight into each protein's function during infection (Specific Aim 2). Finally, the mechanism MVA uses to induce NF-kappa B activation will be defined, mapping the pathways MVA utilizes to induce NF-kappa B activation and the viral proteins responsible (Specific Aim 3). Results obtained here may prove useful in devising novel strategies to overcome pathogenic effects of vaccinia, with implications for enhancing safety and efficacy of a smallpox vaccine.